A hematology analyzer has been usually calibrated before it leaves factory. However, it is possible that its scales of measurement will be drifted when being transported and used, and therefore, the analyzer needs to be calibrated. Calibration parameters of a hematology analyzer include white blood cell (leukocyte, WBC), red blood cell (erythrocyte, RBC) and platelet (PLT) counts, hemoglobin (HGB), mean corpuscular volume (MCV), and hematocrit (HCT). Methods for determining the reference value of these parameters may refer to respective international standard methods. With reference to white blood cells (WBC), according to international reference methods (See, e.g., Clin Lab Haematol., 1994 June; 16(2):131-8), it is only possible to obtain a total white blood cell count, but not possible to achieve leukocyte differentiation. In case of calibrators for various hematology analyzers, it is sufficient that the leukocyte components of calibrators are a single population of granules rather than simulated granules of differential leukocytes (classes of leucytes).
There are many methods of preparing single population of simulated leukocyte granules described in prior art. These methods may be classified as follows: 1) preparing simulated granules of non-cellular origin from pollen, latex particle and the like; 2) treating animal erythrocytes to simulate leukocytes; 3) treating mammalian granulocytes to simulate certain class of leukocytes. These processed single population of simulated leukocyte granules are primarily used to prepare some control products. Although the prior art gives no word to the simulation of leukocyte component for use in calibrators, in fact, the processing methods used are totally applicable to the preparation of simulated leukocyte granules for use in calibrators.
Due to insurmountable defects in cost, compatibility of instrument system and the like, simulated granules of non-cellular origin have been gradually taken over by processed cell-derived granules. For example, based on the original volume of cells, different single populations of simulated leukocyte granules can be obtained by treating animal erythrocytes. U.S. Pat. No. 4,704,364 discloses that human lymphocytes are simulated with human erythrocytes, human monocytes are simulated with turkey erythrocytes, and human granulocytes are simulated with erythrocytes of a shark species. U.S. Pat. No. 5,320,964 discloses that human lymphocytes are simulated with avian nucleated red blood cells, and human monocytes, neutrophils, eosinophils are simulated with red blood cells of reptiles. In accordance with these methods, single population of simulated leukocyte granules are obtained by harvesting animal erythrocytes by centrifugation and fixing them using aldehyde compounds. Such single population of simulated leukocyte granules exhibits good suspensibility and stability, and can be used to simulate leukocyte components for used in control products or calibrators of some hematology analyzers.
There are detailed descriptions for methods of treating mammalian granulocytes in U.S. Pat. No. 6,759,246. By treating granulocytes of swine, cattle and other suitable mammals with reagents such as Bronopol and Triadine-3, granulocytes become similar to human lymphocytes in properties of light scatter, impedance, conductivity and dye uptake, whereby they are identified as lymphocytes on some hematology analyzers. Then, the simulated lymphocytes are fixed using aldehyde reagents as a cell fixative. This patent discloses that compared with human lymphocytes simulated with mammalian lymphocytes, human lymphocyte analogs simulated with granulocytes show greater stability in cell volume on a analyzer. Furthermore, analogs of single population of neutrophils and eosinophils may be also simulated with processed porcine granulocytes, while analogs of single population of monocytes come from processing bovine granulocytes.
However, the prior art methods of preparing analogs of single population of leukocytes still suffer from some drawbacks. When some single population of human leukocyte granules simulated with animal red blood cells are detected on a hematology analyzer using impedance method, a standard histogram distribution of human leukocytes from fresh blood is exhibited (See FIG. 1). Nevertheless, in case of 5-part differential hematology analyzer based on forward light scatter, side light scatter, etc., the scatter plot exhibited by single population of leukocytes which is simulated with animal nucleated red blood cells does not show the distributional characteristics of single population clusters (See FIG. 2), because animal nucleated red blood cells generally have an ellipse rather than a spherical shape. Cells are passed through flow chamber in various “postures”, resulting in nonuniform light scatter properties, for example, sector trailing distribution or complete dispersionas shown in FIG. 2. Although lymphocytes simulated with mammalian granulocytes or other single population of leukocytes can be used for 5-part differential hematology analyzer based on multi-angle light scatter, it requires granulocytes being separated, for example, by separation methods such as density gradient, and moreover special treatment and prolonged fixation. Therefore, these methods are not desirable due to complexity.
To overcome the poor clustering subjected to the leukocyte granules simulated with animal red blood cells, and complexity in processing regarding the single population of leukocytes simulated with animal granulocytes, the present invention provides a method of preparing single population of simulated leukocyte granules, adapted for calibrators of a hematology analyzer employing a multi-angle light scatter theory (e.g., 5-part differential hematology analyzer), which method is simple in operation, has low cost and good granule clustering, and exhibit excellent stability.